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Notes
CSIR - NET

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MODULE

13
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FIRST EDITION REVISED
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Methods in Biology
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®
Editors
Dr. Aditya Arya
Dr. Amit Kumar
 Online
Supplement

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Module 13

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Methods in Biology
Free Online Supplement
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Editors
Aditya Arya, Ph.D.
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Amit Kumar, Ph.D.

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Drawing Pin Publishing

New Delhi
CSIR-NET PrepnoteTM, Module 13 – Online supplement 1

Drawing Pin Publishing, New Delhi, India

Online supplement for Prep-Note CSIR-NET Module 13 - Methods in Biology

First Edition – 10th Oct 2019

ASIN: B07B3SWP6C

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Copyright © 2018. Drawing Pin Publishing. All rights reserved.
All rights reserved. No part of this publication including text, tables and illustrations may be
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photocopying, recording, or any other storage and retrieval system, without prior written
permission of the publisher. All the copyright related queries may be directly sent to the
publisher at [email protected].

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Disclaimer

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Although, utmost care has been taken to avoid any errors in the preparation of answers, however
in case of any discrepancies or loss of any kind due to incorrect answers or solutions, authors or
publishers shall not be responsible

Value creativity and hard work

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At drawing pin publishing, we value the efforts of all team members and therefore provide you a
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APPENDIX

Contents
Part A- Additional Methods – Quick Review
1
S.No. Technique Application Page Online
1 DNase foot printing assay DNA-protein interactions 3
2 Chromosome Walking Genome mapping 4

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3 EMSA DNA-protein interactions 5
4 Bisulphite Sequencing Epigenetics, methylation 6
5 TILLING assay Reverse genetics 7
6 Scanning Tunneling Microscopy Surface analysis 8
7 Atomic force microscopy Surface analysis 9
8 Cryo-electron microscopy Bimolecular structures 10

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9 FRET Bimolecular interactions 11
10 FRAP Biomolecular mobility 12
11 Yeast two Hybrid Bimolecular interactions 13
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Phage display
SELEX
Electroporation
Gene Gun
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Ligand selection
Directed evolution
DNA delivery
DNA delivery
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Part B- Coloured Images of Module 13

1 Chapter A Images 18
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2 Chapter B Images 19
3 Chapter C images 25
4 Chapter D images 28
5 Chapter E images --
6 Chapter F images 30
7 Chapter G images 32
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8 Chapter H images 35

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PART A
Additional
Methods

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MOLDULE 13:
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Methods in Biology
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Aditya Arya, Amit Kumar

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1. DNase Footprinting
A DNase foot printing assay, also known as Deoxyribonuclease I (DNase I) protection mapping, or foot
printing, is a molecular and biochemical method that detects DNA-protein interaction. It was developed by
David Galas and Albert Schmitz at Geneva in 1977. DNase foot printing is based on the fact that, a protein
bound to DNA will remain protected from enzymatic cleavage (DNase). This makes it possible to locate a
protein binding site on a particular DNA molecule. Basic procedure involves digestion of a protein bound DNA
by deoxyribonuclease (DNase) to cut the radioactively end-labeled DNA, followed by gel electrophoresis to
detect the resulting cleavage pattern. A control DNA unbound to protein is used to map the bands missing
in protected DNA.

The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free

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DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein. If the protein
binds DNA, the binding site is protected from enzymatic cleavage. This protection will result in a clear area
on the gel which is referred to as the "footprint".

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Fig. Overall procedure and actual autoradiograph (Image credit - Tomoaki Sakamoto et al, 2001)

Footprinting has been developed further as a quantitative technique to determine separate binding curves
for each individual protein-binding site on the DNA. For each binding site, the total energy of binding is
determined directly from that site's binding curve. For sites that interact cooperatively, simultaneous
numerical analysis of all the binding curves can be used to resolve both the intrinsic binding and cooperative
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components of these energies.

Steps involved: Preparation of a singly end-labeled DNA restriction fragment - equilibration of the protein
with DNA - exposure of the equilibrium mixture to DNase I - electrophoretic separation on gels of the
denatured hydrolysis products – autoradiography.

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Reference and further reading

Galas DJ, Schmitz A (Sep 1978). "DNAse footprinting: a simple method for the detection of protein-DNA binding
specificity". Nucleic Acids Research. 5 (9): 3157–70. – Original paper of inventors.

Brenowitz M, Senear DF, Kingston RE. DNase I footprint analysis of protein-DNA binding. Curr Protoc Mol Biol. 2001
May;Chapter 12:Unit 12.4.

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2. Chromosome Walking
Chromosome walking is a technique using which an unknown region of a chromosome can be explored. It is
generally used to isolate a locus of interest for which no probe is available but that is known to be linked to
a gene which has been identified and cloned. Chromosome Walking was developed by Welcome Bender,
Pierre Spierer, and David S. Hogness in the Early 1980's. In this technique, a fragment containing a known
gene is selected and used as a probe to identify other overlapping fragments which contain the same gene.
The nucleotide sequences of these fragments can then be characterized. This process continues for the
length of the chromosome.

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Fig. Illustration of chromosome walking
The procedure of chromosome walking includes several steps. First step is the formation of the genomic
library selection of a clone of interest (identified by a probe) and sub clone a small fragment from one end
of the clone (there is a technique available to subclone a fragment from the end). In the next step the
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subcloned fragment of the selected clone are hybridized with other clones in the library and a second clone
hybridizing with the subclone of the first clone is identified due to presence of overlapping region. In the
third step, the end of the second clone is then subcloned and used for hybridization with other clones to
identify a third clone having overlapping region with the subcloned end of the second clone. Subsequently,
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third clone identified as above is also subcloned and hybridized with clones in the same manner and the
procedure may be continued. Finally, a restriction map of each selected clone is prepared and compared to
know the regions of overlapping. This generates the order of fragments or clones which contain the DNA
region one after the other. Finally sequencing results are integrated to achieve the complete DNA sequence
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of a chromosome.

There is a limitation to the speed of chromosome walking because of the small size of the fragments that
are to be cloned. Another limitation is the difficulty of walking through the repeated sequence that are
scattered through the gene. If the markers were too far away, it simply was not a viable option. Additionally,
chromosome walking could easily be inhibited by unclonable sections of DNA. A solution to this problem was
achieved with the advent of chromosome jumping (Marx, 1989), which allows the skipping of unclonable
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sections of DNA.

Primer walking is a sequencing method of choice for sequencing DNA fragments between 1.3 and
7 kilobases. It is similar to chromosome walking with a difference that PCR is used instead of cloning a
hybridization approach.

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Reference and further reading

Bender W, Spierer P, Hogness DS, Chambon P. Chromosomal walking and jumping to isolate DNA from the Ace and
rosy loci and the bithorax complex in Drosophila melanogaster (1983). Journal of Molecular Biology.Volume 168 (1),
17-33.

Collins FS, Drumm ML, Cole JL, Lockwood WK, Vande Woude GF, Iannuzzi MC. Construction of a general human
chromosome jumping library, with application to cystic fibrosis. Science. 1987 Feb 27;235(4792):1046-9.

https://journals.plos.org/plosone/browse/chromosome_walking - Image credit

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3. Electrophoretic Mobility Shift Assay (EMSA)

The gel electrophoresis mobility shift assay (EMSA), also known as or mobility shift electrophoresis, also
referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a molecular
technique that is used to detect protein complexes with nucleic acids. The electrophoretic mobility shift assay
(EMSA) is a rapid and sensitive method to detect protein-nucleic acid interactions. It is based on the
observation that the electrophoretic mobility of a protein-nucleic acid complex is typically less than that of
the free nucleic acid. Mobility-shift assays are often used for qualitative purposes, although under appropriate
conditions they can provide quantitative data for the determination of binding stoichiometries, affinities and
kinetics. Most current methods are based on the procedure suggested by Garner MM, Revzin in 1981.

It is the core technology underlying a wide range of qualitative and quantitative analyses for the

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characterization of interacting systems. Most commonly EMSA is used to detect the ability of a known protein
to bind with given part of DNA, such as whether a transcription factor binds to a given promoter or not,
however a large number of variants exist, which are used for various other purposes like time course EMSA
for measurement of association and/or dissociation kinetics, Double-label assays for stochchiometry, Circular
permutation for DNA bending, reverse EMSA for Detection of nucleic acid binding, measurement of binding
affinity, RNA-DNA and RNA-DNA EMSA for non-protein interactions.

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Fig. Illustration showing EMSA, and typical results of EMSA (image credit – Thermo scientific)

Procedure: Solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to
electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the
distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic
acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic
acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic
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mobilities of complexes under assay conditions.

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Reference and further reading

Hellman LM, Fried MG. Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat
Protoc. 2007; 2(8):1849-61.

Garner MM, Revzin A (July 1981). "A gel electrophoresis method for quantifying the binding of proteins to specific DNA
regions: application to components of the Escherichia coli lactose operon regulatory system". Nucleic Acids Res. 9
(13): 3047–60

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4. Bisulphite Sequencing
DNA methylation is an epigenetic mechanism known to play a role in mammalian gene regulation, genomic
imprinting, and suppression of transposable elements. Methylation patterns can be characterized by
interrogating specific loci or through genome-wide methylation profiling. Bisulfite treatment is considered the
gold standard method for determining DNA methylation.

In this method, treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-
methylcytosine residues unaffected. Therefore, DNA that has been treated with bisulfite retains only
methylated cytosines. Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend
on the methylation status of individual cytosine residues, yielding single-nucleotide resolution information
about the methylation status of a segment of DNA. Various analyses can be performed on the altered

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sequence to retrieve this information. The objective of this analysis is therefore reduced to differentiating
between single nucleotide polymorphisms (cytosines and thymidine) resulting from bisulfite conversion.

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Fig. Use of methyl-specific primers and various formats of bisulphite sequencing.
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The first reported method of methylation analysis using bisulfite-treated DNA utilized PCR and standard
dideoxynucleotide DNA sequencing to directly determine the nucleotides resistant to bisulfite conversion.
PCR amplification of bisulfite-treated DNA requires an enzyme capable of tolerating uracil-containing DNA
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and high AT targets (conversion of unmodified cytosines to thymines increases the AT ratio of the target
DNA). Now-a-days, much advanced version have evolved, such as pyrosequencing (a variant of next
generation sequencing) has also been used to analyze bisulfite-treated DNA without using methylation-
specific PCR. Another method called methylation-sensitive single-strand conformation analysis (MS-SSCA)is
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used that involves, single-strand conformation polymorphism analysis (SSCA). SSCA differentiates between
single-stranded DNA fragments of identical size but distinct sequence based on differential migration in non-
denaturating electrophoresis. A further method to differentiate converted from unconverted bisulfite-treated
DNA is using high-resolution melting analysis (HRM), a quantitative PCR-based technique initially designed
to distinguish SNPs. A recently described method by Ehrich et al. further takes advantage of bisulfite-
conversions by adding a base-specific cleavage step to enhance the information gained from the nucleotide
changes. By first using in vitro transcription of the region of interest into RNA (by adding an RNA polymerase
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promoter site to the PCR primer in the initial amplification),

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Reference and further reading

Fraga MF, Esteller M (September 2002). "DNA methylation: a profile of methods and applications". BioTechniques. 33
(3): 632, 634, 636–49.

El-Maarri O (2003). "Methods: DNA methylation". Adv. Exp. Med. Biol. Advances in Experimental Medicine and Biology.
544: 197–204.

Laird PW (April 2003). "The power and the promise of DNA methylation markers". Nat. Rev. Cancer. 3 (4): 253–66.
doi:10.1038/nrc1045.

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5. TILLING ASSAY – Reverse Genetics

TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to
insertional mutagenesis in Arabidopsis thaliana (McCallum et al. 2000). The application of TILLING makes
the functional analysis of large genomes as well as small genes, which are difficult targets for insertional
mutagenesis TILLING takes advantage of classical mutagenesis, sequence availability and high-throughput
screening for nucleotide polymorphisms in a targeted sequence. It combines the high frequency of mutations
induced by traditional mutagenesis with sensitive techniques for discovering single nucleotide mutations. The
main advantage of TILLING as a reverse genetics strategy is that it can be applied to any plant species,
regardless of its genome size, ploidy level or method of propagation.

Chemical mutagens, which are usually used in TILLING protocols, provide a high frequency of point mutations

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distributed randomly in the genome. An analysis of mutations induced by ethyl methanesulphonate (EMS) in
192 Arabidopsis genes revealed about ten mutations per gene among the 3,000 M2 plants examined (Greene
et al. 2003). It was estimated that each M2 plant carried, on average, 720 mutations (Till et al. 2003), while
only 1.5 T-DNA insertions per mutant line were detected in the Arabidopsis insertion populations (Alonso et
al. 2003). Thus, much smaller populations are required to reach saturation mutagenesis using TILLING—ca.
5,000 M1 plants in Arabidopsis (Østergaard and Yanofsky 2004) as compared to 360,000 lines in T-DNA
mutagenesis (Alonso and Ecker 2006). Another great advantage of TILLING technology relies on the ability

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of chemical mutagens to create a spectrum of mutations, including missense changes, truncation and
mutations in splice junction sequences. In contrast to insertional mutagenesis that generates mostly gene
knock-outs, using TILLING, it is possible to induce a series of alleles in a targeted locus. In addition to loss-
of-function alleles, chemical mutagens generate gain-of-function and hypomorphic alleles that can provide a

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range of phenotypes (Alonso and Ecker 2006). The mutations are stable, which is not always the case for
alternative methods of reverse genetics utilising RNAi silencing or transposon, e.g. Ac/Ds tagging. In addition,
RNAi technology and insertional mutagenesis through T-DNA or transposon tagging relies on genetic
transformation. TILLING does not require transformation and, thus, is the only reverse genetics strategy
applicable for species that are not transformable or recalcitrant. It is recommended as non-GMO technology,
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so when using TILLING, GMO procedures and controversies are avoided. Moreover, TILLING is not technically
demanding and can be performed at a relatively low cost.

Some publicly accessible databases may constitute independent websites, whose only purpose is to describe
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either the specific project (LycoTILL, http://www.agrobios.it/tilling) or several projects carried out by one
team (UTILLdb, http://urgv.evry.inra.fr/UTILLdb). When creating meta-services involving a number of
different databases that already exist on various aspects of studies on one organism, TILLING databases can
also be included (MaizeGBD, http://www.maizegdb.org/).
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Fig. Outline procedure of TILLING assay.

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Reference and further reading

Kurowska M, Daszkowska-Golec A, Gruszka D, et al. TILLING: a shortcut in functional genomics. J Appl Genet.
2011;52(4):371-90.

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6. Scanning Tunnelling Microscopy

The development of the family of scanning probe microscopes started with the original invention of the STM
in 1981. Gerd Binnig and Heinrich Rohrer developed the first working STM while working at IBM Zurich
Research Laboratories in Switzerland, for which they were awarded Nobel prize in physics in 1986.

The STM is based on the principle of quantum mechanical effect called tunneling between a sharp tip and
surface of object at atomic level. It is this effect that allows us to “see” the surface using a sharp tip. The
tunneling from tip to surface with the tip rastering with piezoelectric positioning, with the feedback loop
maintaining a current setpoint is used to generate a 3D image of the electronic topography.

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Fig. Outline of scanning tunneling microscopy and its principle, sample image (credit – hiffman.physcis)

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In order to understand the principle of STM, we must first know the meaning of tunnelling current. Tunneling
is a quantum mechanical effect. The electrons in the tip and the sample are considered to be in two separate
energy valleys, separated by a hill which is the vacuum barrier, but the moment when a bias voltage is
applied to the sample with respect to the tip, the Fermi level of the sample is effectively raised with respect
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to the tip. When a bias voltage Vb is applied between tip and sample, a current will flow, and this current
can be measured as a function of (x, y) location and as a function of Vb. This results in an empty states
available for tunneling of electrons into the tip resulting in the flow of current. The current which flows
between the tip and the sample depends on the voltage difference between the tip and sample. This voltage
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is used for obtaining image of the surface.

Next important feature of STM is the maintenance of tip at specific position which is critical to imaging the
object at ultra-high resolution, it is regulated by two physical phenomenon, piezoelectric effect and feedback
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loop. The piezoelectric effect was discovered by Pierre Curie in 1880. The effect is created by squeezing the
sides of certain crystals, such as quartz or barium titanate. The result is the creation of opposite charges on
the sides. The effect can be reversed as well; by applying a voltage across a piezoelectric crystal, it will
elongate or compress. These materials are used to scan the tip in an scanning tunneling microscopy (STM)
and most other scanning probe techniques. A typical piezoelectric material used in scanning probe
microscopy is PZT (lead zirconium titanate). Feedback loop is another essential factor needed in electronic
to measure the current, scan the tip, and translate this information into a form that we can use for STM
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imaging. A feedback loop constantly monitors the tunneling current and makes adjustments to the tip to
maintain a constant tunneling current. These adjustments are recorded by the computer and presented as
an image in the STM software. Such a setup is called a constant current image.

There is a major drawback of scanning tunneling microscopy is that it can only image conducting or
semiconducting surfaces. The atomic force microscope (AFM) was developed to overcome this basic
drawback and allow the imaging of almost any type of surface, including polymers, ceramics, composites,
glass, and biological samples.

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Reference and further reading

http://hoffman.physics.harvard.edu/research/STMintro.php
http://hoffman.physics.harvard.edu/research/STMtechnical.php - Technical details of STM

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7. Atomic Force Microscopy

Atomic force microscopy (AFM) was invented by Binnig, Quate, and Gerber invented the AFM in 1985. Their
original AFM consisted of a diamond shard attached to a strip of gold foil. Modern AFM tips (or probes) and
cantilevers are typically micro-fabricated from Si or Si3N4. Normally, the probe is a sharp tip, which is a 3-6
um tall pyramid with 15-40 nm end radius.

The name AFM, is based on the fact that this microscopy relies on the forces between the tip and sample.
The force is not measured directly, but calculated by measuring the deflection of the lever, knowing the
stiffness of the cantilever (using Hookes’ law; F = -kz, where F is the force, k is the stiffness of the lever,
and z is the distance the lever is bent), operate by measuring force between a probe and the sample. To
acquire the image resolution, AFMs can generally measure the vertical and lateral deflections of the cantilever

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by using the optical lever. The optical lever operates by reflecting a laser beam off the cantilever. The
reflected laser beam strikes a position-sensitive photo-detector consisting of four-segment photo-detector.
The differences between the segments of photo-detector of signals indicate the position of the laser spot on
the detector and thus the angular deflections of the cantilever.

Similar to STM, the position of tip in AFM is controlled by feedback loop control. The feedback loop consists
of the tube scanner that controls the height of the tip; the cantilever and optical lever, which measures the

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local height of the sample; and a feedback circuit that attempts to keep the cantilever deflection constant by
adjusting the voltage applied to the scanner.

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Fig. AFM principle and an image of lambda DNA acquired by AFM (credit – M. Guthold)

AFM can work in two different modes, contact mode and non-contact mode. In contact mode, the tip is
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"dragged" across the surface of the sample and the contours of the surface are measured either using the
deflection of the cantilever directly or, more commonly, using the feedback signal. In non-contact atomic
force microscopy mode, the tip of the cantilever does not contact the sample surface. The cantilever is
instead oscillated at either its resonant frequency (frequency modulation), the decrease in frequency is
measured as a result of van der Waals forces or any other long-range force that extends above the surface.
(Non-contact mode does not suffer from tip or sample degradation hence useful for measuring soft samples,
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e.g. biological samples).

AFM has application in solid state physics for the identification of atoms at a surface, in molecular biology,
to study the structure and mechanical properties of protein and nucleic acid complexes. In cellular biology,
AFM can be used to attempt to distinguish cancer cells and normal cells based on a hardness of cells, and to
evaluate interactions.

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Reference and further reading

https://www.nanoscience.com/techniques/atomic-force-microscopy/

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8. Cyro-Electron Microscopy
Traditionally, X-ray crystallography and NMR spectroscopy represent major workhorses of structural
biologists, with the lion share of protein structures reported in protein data bank (PDB) being generated by
these powerful techniques. Despite their wide utilization in protein structure determination, these two
techniques have logical limitations, with X-ray crystallography being unsuitable for the analysis of highly
dynamic structures and with NMR spectroscopy being restricted to the analysis of relatively small proteins.
EM allows the direct visualization and 3D reconstruction of individual purified molecules or complexes of
molecular mass greater than ∼100 kDa, isolated assemblies, tissue sections, organelles or even whole cells,
provided that they are thin enough (<1 μM) to transmit the electron beam.

Cryogenic Electron Microscopy (cryoEM) is an advanced version of conventional electron microscopy

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optimised for preserving the biological structures and excluding the requirement of sample fixing, so as to
visualize the biomolecules such as proteins. Jacques Dubochet, Joachim Frank, and Richard Henderson were
awarded Nobel Prize in chemistry in the year 2017, for the development of cryo-electron microscopy for the
high-resolution structure determination of biomolecules in solution. In this method, a small aliquot of sample
in solution or suspension is applied to an electron microscopy grid and is blotted to a thin layer and
immediately plunged into liquid ethane (around −180 °C), so that the molecules get trapped in a layer of
vitrified water. Ideally the ice should be just thick enough to accommodate the particles without distortion.

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Because of their high sensitivity to electron damage, biological cryo-EM samples must be imaged under low
dose conditions, with an electron dose of 10 –20 e−Å2 to preserve high-resolution details. Following the cryo-
vitrification samples are stained by negative staining approach that helps to clearly visualize the sample and
check its homogeneity. In cryo-EM, the vitrified sample is imaged by collecting movie frames that are aligned

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for motion correction and then averaged. Particles are boxed from the micrographs, centered and aligned.
Classification and averaging give improved signal to noise ratio (SNR), and class averages can be used to
obtain a low-resolution initial model by common lines or tilt methods. After alignment, classification and
cleaning of the dataset, particles are assigned orientations by projection matching to the initial model.
Orientation refinement is performed iteratively until the structure converges.
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Figure. Outline procedure of sample vitrification and cryo electron microscopy.

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Reference and further reading

Carroni M, Saibil HR. Cryo electron microscopy to determine the structure of macromolecular complexes. Methods.
2016;95:78‐85. doi:10.1016/j.ymeth.2015.11.023.

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9. Foster Resonance Energy Transfer (FRET)

Förster Resonance Energy Transfer (FRET) is a microscopy based technique based on a physical
phenomenon whereby energy created by fluorescence excitation of one molecule is transferred to an
adjacent molecule. This is also called as Fluorescence Resonance Energy Transfer. Förster resonance
energy transfer is named after the German scientist Theodor Förster.

This technique is based is on the fact that when two fluorophore are used and the excitation of the first
molecule (Donor) results in fluorescence emission of the second molecule (Acceptor). Under typical
fluorescence conditions relaxation of the electron energy would result in the emission of a photon
(fluorescence) by the donor fluorophore, with a resultant drop in molecule energy. However, if a
suitable acceptor molecule is within a certain distance (Förster distance; <10nm) then donor energy can

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experience transfer of energy (Kr) to the acceptor, resulting in acceptor fluorescence. An important concept
is that the transfer of energy between molecules is non-radiative, and that the distances involved are much
less than the wavelength of light. Föster energy transfer is therefore distinguished from a more common
radiative transfer of energy (e.g., absorption of the Ex photon by a molecule.).

This technique is used to study the protein-protein interactions both in vivo and in vitro. Two potential
proteins are conjugated to donor and acceptor fluorophore and the change in fluorescence is observe, if they

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interact, FRET occurs otherwise not. A prime requirement for FRET is that two molecules must be
less than 10 nm apart and donor emission must overlap acceptor excitation therefore cyan and
yellow fluorescent protein combination is considered best. FRET has been used to detect the location
and interactions of genes and cellular structures including integrins and membrane proteins. FRET can be

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used to obtain information about metabolic or signaling pathways. FRET is also used to study lipid rafts in
cell membranes. FRET is also the common tools in the study of biochemical reaction kinetics and molecular
motors.
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Fig. Illustration of FRET and its application in protein-protein interaction

It should also be noted that FRET is not restricted to fluorescence. It can occur in connection with
phosphorescence as well. Related techniques include BRET, Bioluminescence Resonance Energy Transfer,
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where the donor emits light through luminescence (typically Luciferase) and acceptor.

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Reference and further reading

Kenworthy AK. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy.
Methods. 2001 Jul;24(3):289-96.

Margineanu A, Chan JJ, Kelly DJ, et al. Screening for protein-protein interactions using Förster resonance energy
transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM). Sci Rep. 2016;6:28186. Published 2016 Jun 24.

Truong, Kevin; Ikura, Mitsuhiko (2001). "The use of FRET imaging microscopy to detect protein–protein interactions and
protein conformational changes in vivo". Current Opinion in Structural Biology. 11 (5): 573–8.

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10. Fluorescence Recovery after Photobleaching (FRAP)

Fluorescence recovery after photobleaching (FRAP) is a technique used to quantify diffusion. Although
developed in the 1970s, the discovery and further development of fluorescent proteins revolutionised
FRAP. After the discovery of green fluorescence protein and its application as a noninvasive and genetically
coded protein‐tag, in vivo studies of protein dynamics and interactions became possible.
In this method a lipid or a protein (whose diffusibility is to be checked) is tagged with a fluorophore.
Tagging can be done ex situ (by conjugation of fluorophore) or in situ (by preparing FP tagged
recombinant fusion proteins). A strong beam of laser falling over fluorescent are can abolish the
fluorescence, this is known as photobleaching. Now, if the molecules do not diffuse, the bleached part
will remain fluorescence-less for ever, but if the molecules diffuse it will regain its fluorescence gradually
due to movement of molecules from adjoining areas, this is known as recovery. The time of recovery is

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proportional to the rate of diffusion, so it can be used to compare diffusion rates of different molecules.
Subsequent time-lapse imaging is performed and the rate of recovery as well as the mobile fraction can
be quantified.
FRAP can be conducted on proteins diffusing in 2D, for example in the plane of the plasma membrane, or
3D, such as in the cytosol, and appropriate 2D or 3D diffusion coefficients can subsequently be
determined. FRAP experiments are often conducted on confocal microscopes. To derive quantitative
results from such experiments, several parameters and controls need to be considered and utilised in the

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analysis.

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Fig. Illustration of FRET and its application in protein-protein interaction

Among various types of cell components, the most fluidic are membrane lipids, hence they are expected to
have fastest recovery or smaller recovery time. This is usually followed by surface attached proteins.
However, FRAP experiments on transmembrane proteins would show much slower recovery and any
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transmembrane-protein anchored to cytosolic proteins will have least diffusion rate and hence slowest
recovery.

The FRAP experiment itself is divided into three separate phases, first the pre‐bleach phase, second
bleaching, and third post‐bleach recovery. The pre‐bleach imaging phase is intended to provide readout of
the initial fluorescence within the observation volume, and to establish that the biological and experimental
systems are stable. The bleach phase uses intense excitation light to bring about localized photobleaching.
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In the post‐bleach phase, fluorescence recovery within the bleached region is monitored.

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Reference and further reading

Pincet F, Adrien V, Yang R, Delacotte J, Rothman JE, et al. (2016) FRAP to Characterize Molecular Diffusion and
Interaction in Various Membrane Environments. PLOS ONE 11(7).

Staras K, Mikulincer D, Gitler D. Monitoring and quantifying dynamic physiological processes in live neurons using

fluorescence recovery after photobleaching. J Neurochem. 2013 Jul; 126(2):213-22.

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11. Yeast two hybrid System (Y2H)

Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique
used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical
interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.
This technique was pioneered by Stanley Fields and Ok-Kyu Song in 1989, the technique was originally
designed to detect protein–protein interactions using the Gal4 transcriptional activator of the yeast
Saccharomyces cerevisiae. The underlying principle of Y2H is reconstitution of a functional transcription
factor (TF) when two proteins or polypeptides of interest interact.

In order to achieve reconstitution of a promoter, two different domains of a transcription factor, one binding
with DNA (DNA binding domain or DBD) and other interacting with RNA pol (activation domain) are fused to

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two separate protein in question (Infact to their genes by recombinant DNA technology) respectively.
Therefore, two fusions (‘hybrids’) are constructed between each protein of interest and either the DNA
Binding Domain (DBD) or the Activation Domain (AD) of the TF. The protein fused to the DBD is referred to
as the ‘bait’, and the protein fused to the AD as the ‘prey’. Upon interaction between the bait and the prey,
the DBD and AD are brought in close proximity and a functional TF is reconstituted upstream of the reporter
gene. If the two proteins don’t interact, this does not lead to complete formation of a transcriptional activator
and hence, target gene is not expressed (reporter). The most popular fusions use the DBD and AD of the

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yeast TF Gal4. The bacterial protein LexA is also frequently used as a DBD in combination with Gal4 AD. The
most popular reporter genes are HIS3 to select yeast on a medium lacking histidine, and LacZ to screen
yeast in a colorimetric assay.

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Fig. Principle involved in yeast two hybrid, vectors used for constructing bait and prey, and representative figure showing Lac Z
based selection of positive colonies (in which interaction of proteins in question occurred).

Variations of the yeast two-hybrid were developed to conduct screens in the presence of a co-factor, or an
enzyme required for a given post-translational modification of the protein partners. Other versions allow to
screen integral membrane proteins and the technique was adapted to detect protein-protein interactions in
mammalian cells. Finally, yeast n-hybrid protocols were also devised to screen for novel DNA-protein, RNA-
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protein6 and small molecule-protein interactions. RNA-protein interactions can be studied through a three-
hybrid variation of the two-hybrid technique. In this case, a hybrid RNA molecule serves to adjoin together
the two protein fusion domains—which are not intended to interact with each other but rather the
intermediary RNA molecule (through their RNA-binding domains). Simultaneous detection of protein–protein
and protein–DNA interaction is done by its variant called one-two-hybrid approach.

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Reference and further reading

Fields’ S, Song O (July 1989). "A novel genetic system to detect protein-protein interactions" (abstract). Nature. 340
(6230): 245–6.

Luo Y, Batalao A, Zhou H, Zhu L (February 1997). "Mammalian two-hybrid system: a complementary approach to the
yeast two-hybrid system" (PDF). Bio Techniques. 22 (2): 350–2

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12. Phage display

Phage display technology is an in vitro screening technique for identifying ligands for proteins and other
macromolecules. At the crux of phage display technology is the ability to express peptide or protein
sequences as fusions to the coat proteins of a bacteriophage. Libraries of phage-displayed peptides or
proteins are thereby physically linked to their encoding nucleic acid, allowing selection of binding partners
for myriad target types by iterative rounds of in vitro panning and amplification, followed by DNA sequencing.
Libraries of over a billion members can be screened in a matter of days, offering an efficient alternative to
more traditional methods of epitope mapping, receptor ligand identification, or protein evolution. Phage
display process involve few simple steps. Step 1. fusion proteins for a viral coat protein + the gene to be
evolved (typically an antibody fragment) are expressed in bacteriophage. Step 2. The library of phage are

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washed over an immobilised target. Step 3. The remaining high-affinity binders are used to infect bacteria.
4) the genes encoding the high-affinity binders are isolated. Step 5. those genes may have random mutations
introduced and used to perform another round of evolution. The selection and amplification steps can be
performed multiple times at greater stringency to isolate higher-affinity binders.

Applications of phage display technology include determination of interaction partners of a protein (which
would be used as the immobilised phage "bait" with a DNA library consisting of all coding sequences of a
cell, tissue or organism) so that the function or the mechanism of the function of that protein may be

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determined. Phage display is also a widely used method for in vitro protein evolution (also called protein
engineering). As such, phage display is a useful tool in drug discovery. It is used for finding new ligands
(enzyme inhibitors, receptor agonists and antagonists) to target proteins. The technique is also used to
determine tumour antigens (for use in diagnosis and therapeutic targeting) and in searching for protein-DNA

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interactions using specially-constructed DNA libraries with randomised segments. Recently, phage display
has also been used in the context of cancer treatments - such as the adoptive cell transfer approach. In
these cases, phage display is used to create and select synthetic antibodies that target tumour surface
proteins. These are made into synthetic receptors for T-Cells collected from the patient that are used to
combat the disease
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Fig. Typical phage display cycle.

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Reference and further reading

Smith GP, Petrenko VA. Phage Display. Chem Rev. 1997;97(2):391‐410. doi:10.1021/cr960065dLuo Y, Batalao A, Zhou
H, Zhu L (February 1997). "Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid
system" (PDF). Bio Techniques. 22 (2): 350–2.

Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface.
Science. 1985;228(4705):1315‐1317.

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13. SELEX
Systematic evolution of ligands by exponential enrichment (SELEX), also referred to as in vitro selection or
in vitro evolution, is a combinatorial chemistry technique in molecular biology for producing oligonucleotides
of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. Although SELEX has
emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB
(selected and amplified binding site) and CASTing (cyclic amplification and selection of targets). The process
begins with the synthesis of a very large oligonucleotide library consisting of randomly generated sequences
of fixed length flanked by constant 5' and 3' ends that serve as primers. For a randomly generated region of
length n, the number of possible sequences in the library is 4n (n positions with four possibilities (A,T,C,G)
at each position). The sequences in the library are exposed to the target ligand - which may be a protein or

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a small organic compound - and those that do not bind the target are removed, usually by affinity
chromatography. The bound sequences are eluted and amplified by PCR to prepare for subsequent rounds
of selection in which the stringency of the elution conditions is increased to identify the tightest-binding
sequences. An advancement on the original method allows an RNA library to omit the constant primer
regions, which can be difficult to remove after the selection process because they stabilize secondary
structures that are unstable when formed by the random region alone.

SELEX has been used to develop a number of aptamers that bind targets interesting for both clinical and

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research purposes. Also towards these ends, a number of nucleotides with chemically modified sugars and
bases have been incorporated into SELEX reactions.These modified nucleotides allow for the selection of
aptamers with novel binding properties and potentially improved stability.

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Fig. The typical SELEX cycle (Image credit : Lewis Fraser et. al, IJMS, 2017, 18(12):2516)

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Reference and further reading

C TUERK, L GOLD., Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA
polymerase. Science, 03 AUG 1990 : 505-510.

Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346(6287):818‐
822.

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14. Electroporation
Electroporation, electro transfer or electropermeabilization, is a method of DNA transfer often used in genetic
engineering, in which an electrical field is applied to cells in order to increase the permeability of the cell
membrane, allowing the transfer of foreign DNA into the host cell. Besides genetic engineering this method
can also be used for the transfer of small molecules, drugs etc. into the cell. This method can be used with
most cell types, yields a high frequency of both stable transformation and transient gene expression, and,
because it requires fewer steps, can be easier than alternate techniques. The underlying principle of
electroporation is simple and based on creation of nm-scale water-filled holes in the membrane. During
electroporation the lipid molecules are not chemically altered but simply shift position, opening up a pore
which acts as the conductive pathway through the bilayer as it is filled with water. Upon application of this

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potential the membrane charges like a capacitor through the migration of ions from the surrounding solution.
Once the critical field is achieved there is a rapid localized rearrangement in lipid morphology. The resulting
structure is believed to be a "pre-pore" since it is not electrically conductive but leads rapidly to the creation
of a conductive pore. Evidence for the existence of such pre-pores comes mostly from the "flickering" of
pores, which suggests a transition between conductive and insulating states. It has been suggested that
these pre-pores are small (~3 Å) hydrophobic defects.

Direct current or DC voltage is used in this method and the field strength is measured as the voltage delivered

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across an electrode gap and is expressed as kV/cm. Field strength is critical to surpassing the electrical
potential of the cell membrane to allow the temporary reversible permeation or “pore formation” to occur in
the cell membrane. Three factors should be considered for optimizing field strength first, cuvette gap size,
2. Cell Diameter (inversely related) and third, Temperature. The distance between electrodes, or “gap size”

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is important when optimizing electroporation experiment. Field strength is calculated using voltage divided
by gap size. For example, using a 4mm gap cuvette with 500V would provide a field strength of 1.25kV/cm.
If instead of a 4mm gap cuvette, a 2mm gap cuvette was used, the voltage would have to be reduced by
half or 250V in order to maintain the same field strength of 1.25kV/cm. The calculation for this is Field
strength (kV) multiplied by gap size (cm) equals voltage. For example, if a user was certain that a 1.25 kV/cm
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field strength was required in a 1mm gap cuvette the calculation would be: 1.25kV x 0.1cm= 0.125kV or
125volts.

Electrofusion is an expansion of electroporation using different buffers and one or more proprietary
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alternating current (AC) pulse(s). Cells are brought together or “aligned” by the use of an AC pulse which
causes charges to form on the cellular membrane (dielectrophoresis) resulting in alignment of cells or pearl-
chain (dimer) formation. Following the AC cellular alignment the DC pulse is applied to induce permeation of
the cell membrane. When cells are brought into contact during electroporation, these cells are induced to
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fuse. Following this DC pulse the AC pulse is maintained to allow complete cell membrane fusion during the
recovery period.
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Fig. Electroporation process and mechanism of pore formation.

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Reference and further reading

Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation
in high electric fields. EMBO J. 1982;1(7):841‐845.

Sugar IP, Neumann E. Stochastic model for electric field-induced membrane pores. Electroporation. Biophys Chem.
1984;19(3):211‐225. doi:10.1016/0301-4622(84)87003-9

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15. Gene Gun

In genetic engineering, a gene gun or biolistic particle delivery system is a device used to deliver exogenous
DNA (transgenes), RNA, or protein to cells. By coating particles of a heavy metal with a gene of interest and
firing these micro-projectiles into cells using mechanical force, an integration of desired genetic information
can be induced into cells. The gene gun was originally a Crosman air pistol modified to fire dense tungsten
particles. It was invented by John C Sanford, Ed Wolf, and Nelson Allen at Cornell University along with Ted
Klein of DuPont between 1983 and 1986. The original target was onions (chosen for their large cell size),
and the device was used to deliver particles coated with a marker gene which would relay a signal if proper
insertion of the DNA transcript occurred.

The technique involves micro-projectile delivery of DNA. The force comes from a compressed, inert gas such

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as helium at a pressure on the order of 200-300 psi. This device is able to transform almost any type of
cell and is not limited to the transformation of the nucleus; it can also transform organelles, including plastids
and mitochondria. Gene guns can be used effectively on most cells but are mainly used on plant cells. The
procedure includes a few common steps. In step 1 DNA to be transferred is coated with gold or tungsten
particles. The metal particles are usually 0.45–1.5 μm in diameter. In the second step the gene gun
apparatus is made ready to fire. In third step Helium fills the chamber and pressure builds against the
rupture disk. In the next step, the pressure eventually reaches the point where the rupture disk breaks, and

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the resulting burst of helium propels the DNA/gold-coated macro carrier ('Plastic Disk') into the stopping
screen. Finally, in the last step when the macro carrier hits the stopping screen, the DNA-coated gold particles
are propelled through the screen and into the target cells. Because the particles penetrate into the cytosol
and cell nucleus, the efficacy of DNA transduction is substantially higher than that of other approaches and

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therefore substantially reduced doses of DNA plasmid are required (0.1 μg/dose).
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Figure. A typical procedure of gene gun mediated DNA transfer. (Gene Gun image: WT Godbey, 2014)

The gene gun is limited to exposed tissues, such as the epidermis, or cells in a plant leaf. Only limited
penetration is attainable with the gene gun (100-500 μm), so it would be extremely difficult to transfect cells
of an entire organ with this method. The gene gun system has a number of advantages in relation to the
more common needle injection system. It facilitates direct transfer of the gene in various tissues, overcoming
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barriers such as the cell wall, and this gene transfer is not affected by the kinds of cell, receptors, and
molecules at the cell surface. Furthermore, degradation of the gene is reduced, as they are forced to enter
the cytoplasm of target cells and bypass the enzymes in endosomes and lysosomes, and nuclear hurdles. In
addition, the gene gun allows delivery of several genes simultaneously to the same target tissues, which
enables studying the interaction among the gene products. Because the gene gun delivers a small amount
of DNA efficiently, it is a promising approach for DNA vaccination.

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Reference and further reading

Klein, T.M.; Wolf, E.D.; Wu, R.; Sanford, J.C. (May 1987). "High-velocity microprojectiles for delivering nucleic acids into
living cells". Nature. 327 (6117): 70–73.

Benfey, P. N.; Chua, N.-H. (1990-11-16). "The Cauliflower Mosaic Virus 35S Promoter: Combinatorial Regulation of
Transcription in Plants". Science. 250 (4983): 959–966.

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PART B
COLOURED
IMGAGES

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MOLDULE 13:
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Methods in Biology
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Aditya Arya, Amit Kumar

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Chapter A FIGURES

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Chapter B FIGURES
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Chapter C FIGURES
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Chapter D FIGURES
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Figure D14

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Chapter F FIGURES
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Figure F4

Image Credit : Nikon

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Chapter G FIGURES
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Chapter H FIGURES
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